Work by Sir Joshua Reynolds was among those uploaded to Wikipedia
The National Portrait Gallery (NPG) is threatening legal action after 3,300 images from its website were uploaded to online encyclopaedia Wikipedia.
A contributor to the popular site, Derrick Coetzee, breached English copyright laws by posting images from the gallery's collection, the NPG said.
But photographs of works of art are not protected by copyright in the US, where Mr Coetzee and Wikipedia are based.
The NPG said the breach undermines its £1m project to digitise its collection.
So far, 60,000 hi-resolution photographs of paintings held by the NPG have been added to its website for use by the general public.
Sir Joshua Reynolds' portrait of Georgiana Duchess of Devonshire as a child, and a portrait of Victorian photographer Julia Margaret Cameron were among those uploaded to Wikipedia by Mr Coetzee.
Margaret Arnott, a partner at trade mark lawyers Mathys & Squire, told the BBC that a photograph of a work of art is not copyrighted in the US if it is an exact replication, whereas it is in the UK.
In a statement, the NPG said: "The gallery is very concerned that potential loss of licensing income from the high-resolution files threatens its ability to reinvest in its digitisation programme and so make further images available.
"It is one of the gallery's primary purposes to make as much of the collection available as possible for the public to view.
"Digitisation involves huge costs including research, cataloguing, conservation and highly-skilled photography. Images then need to be made available on the gallery website as part of a structured and authoritative database."
The NPG said it asked Wikipedia to remove the images, but did not get a response, and issued the letter to Mr Coetzee as their "only course of action".
The gallery stressed that it hoped to avoid taking any further legal action.
The NPG said while it would be "happy" for the website to use low-resolution images, Mr Coetzee found a way to get around their software and download hi-resolution images without permission.
Writing on Wikipedia in May, Mr Coetzee said it was "no longer possible to obtain high-resolution images from NPG" in the manner he did.
Mr Coetzee also uploaded the legal letter from the NPG, sent by London solicitors Farrar and Co, to Wikipedia.
He said he reproduced the letter to "enable public discourse on the issue". He added that he was taking legal advice.
Mr Coetzee told the BBC he was unable to comment further at this time.